We are studying the reactions required for the replication of duplex DNA in vitro using proteins encoded by T4 bacteriophage. The T4 gene 41 and T4 gene 61 priming proteins synthesize ribopentanucleotides of sequence pppACNNN which are elongated by the T4 gene 43 DNA polymerase, T4 gene 45 protein, the complex of the T4 gene 44 and T4 gene 62 proteins (a DNA-dependent ATPase), and the T4 gene 32 helix destabilizing protein. The stoichiometry of the priming reaction suggests that it is catalyzed by a complex of an oligomer of 41 protein and a 61 protein monomer. The T4 41 protein is also a DNA helicase which unwinds short fragments annealed to single-stranded DNA in a 3' to 5' direction, and increases the rate of chain elongation during strand displacement synthesis on nicked duplex DNA by T4 DNA polymerase and the T4 44/62, 45, and 32 proteins. The gene 44/62 and 45 polymerase accessory proteins permit the polymerase to bind more tightly to DNA in an ATP-dependent reaction. They thus facilitate the movement of the polymerase through kinetic barriers in the DNA in both the 5' to 3' polymerization and 3' to 5' exonuclease reactions. We are searching for T4 or host proteins required for the initiation of replication of duplex T4 DNA.